1. Field of the Invention
The present invention is directed to an apparatus for determining the presence of microorganisms, chemicals, and other analytes in physiological, biological and environmental specimens. Thus the present invention performs diagnostic tests for pathogenic organisms, detection and identification of toxin and drug contamination in food for human and animal consumption and monitoring for pesticide residues in water, soil and food.
2. Description of the Prior Art
As used in this specification the word "analyte" is a term from analytical chemistry meaning the compound for which an assay is developed (e.g., a mycotoxin, its metabolite, and toxin-DNA adducts are all different analytes that might be detected by different assays).
Almost all physiological, biological and environmental fluids are composed of a liquid phase (solvent) and a non-liquid phase. The non-liquid phase consists of two main constituents: i) insoluble substance (i.e., solids and sediments) such as microorganisms, cellular debris, crystals, and particles; and ii) soluble substances (i.e., solutes) such as organic and inorganic substances.
For detection of any abnormalities and/or contaminants of such fluids, the fluid constituents must be separated or extracted from the liquid phase to allow for specialized testing being performed. This has been accomplished by a series of processes involving a number of different containers and expensive laboratory equipment. Examples of these processes include: separation of insoluble matter using filtration and/or centrifugation; and solid-phase extraction of soluble substances. Once the fluid constituent is isolated from the liquid phase a series of qualitative and/or quantitative tests can be performed to determine the presence or absence of the analyte and measure its concentration in solution.
Examples of these tests include Immunoassays (IA), Solid-Phase Extraction (SPE) using Gas and Liquid Chromatography (GC, LC), Mass Spectrometry (MS), cell culturing on special media, in situ Hybridization using DNA and/or RNA probes, DNA and/or RNA target and signal amplification using Polymerase Chain Reaction (PCR). Mass testing using such a series of processes is expensive, time consuming, and often unsatisfactory.
Specialized Testing using Insoluble Substances
Detection and identification of microorganisms:
The traditional methods of detecting and identifying microorganisms in physiological and biological fluids require cell culturing on laboratory media (sometimes followed by susceptibility testing to determine resistance to a particular antibiotic), identification of the organism by detection of serum antibodies against the organism. These methods may require 24 hours to ten days to perform and therefore do not necessarily contribute toward patient management and diagnosis. In some cases the microorganism cannot be isolated or cultured. The use of cytology cups and membranes to isolate cells or microorganisms from wide variety of fluids is known in the art. The Nuclepore Schisto-Kit TM is designed for rapid and accurate quantification of Schistosome eggs in urine by the membrane filtration technique. A simple syringe filtration permits collection of virtually all eggs onto the smooth flat surface of a transparent Nuclepore polycarbonate membrane filter. Quantitative egg counts without staining are easily made with a low power magnifier. Other cytology cups are marketed under the trademark SWIN-LOCK and Swinnex Disc Filter Holder. Nuclepore polycarbonate membranes are used for diagnostic cytology. The surface allows collection of atypical cells from all types of body fluids. More recently the use of Polymerase Chain Reaction (PCR) as a DNA and/or RNA target and signal amplification system and the use of nucleotide probes coupled with in situ hybridization techniques have opened the door to more sensitive and specific tests for identification of the organism. However, these tests as much as other laboratory tests demand minimal manipulations of the sample being tested to minimize potential sample contaminations and consequently very high rate of false test results.
Specialized Testing using Soluble Substances
A. Solid Phase Extraction
Solid phase extraction has rapidly gained acceptance as an efficient, reproducible sample preparation technique. In solid phase extraction, chromatographic sorbents (usually bonded phases) are used to selectively separate analytes and/or interfering impurities from a sample matrix. Solid phase extraction offers a range of selectivity that cannot be achieved with traditional liquid/liquid extraction techniques. Chromatographic columns have been developed for selective isolation or concentration of interesting components from a complex mixture or a large sample volume. For this solid phase extraction (also known as SPE) one takes advantage of different types of interactions (non-polar, polar, or ion exchange) between the sample components, a solid sorbent and a suitable eluent. By selection of the appropriate sorbent interaction (Polar, Hydrophobic, and Ionic) the analyst can utilize the solid phase to separate a wide range of analytes and/or interfering impurities from the sample matrix. Contrary to conventional liquid-liquid extraction solid phase extraction saves solvent and time. Due to combination of different interactions the method features a high degree of versatility. Additionally, problems such as long times for phase separation or formation of stable emulsions do not occur. In liquid-liquid extraction this can only be achieved by multiple extractions. Besides selective fractionation another application of solid phase extraction is enrichment of substances from dilute solutions for trace determinations, since interesting components can be absorbed from a large sample volume and eluted with a small amount of solvent.
B. Affinity Chromatography
Affinity chromatography is a powerful technique for separating molecules based on specific binding to sites on proteins or other biopolymers. Affinity chromatography can be used for either analytical or preparative separations. The material of interest can be a macromolecule such as a protein, in which case a specific small ligand may be immobilized on the stationary phase matrix to cause retention or the ligand immobilized on the column can be a protein which interacts specifically with another substance, usually a macromolecule, to cause retention. Nucleic acids can also be immobilized, creating the possibility of retention by specific base sequence recognition. Affinity chromatography is ordinarily a simple two-step process. A solution containing the substance of interest is forced through the affinity column under conditions favoring specific interaction. This causes the material of interest to become attached to the column while other substances pass directly through. The column is washed with a solution that weakens the specific binding interaction, and the material of interest is immediately eluted from the column in greatly purified form.
C. Immunoassays
Immunochemical detection is based on the ability of an antibody to act as a receptor for the analyte of interest; binding occurs through ionic and van der Waals forces and is unrelated to properties of volatility, thermal stability, and chromogenicity. The high degree of selectivity in antibody binding allows quantification of trace chemicals. When surveying the wide range of analytical techniques available, the use of immunochemical techniques becomes inevitable for some chemicals, since immunoassays allow measurements not possible by other means. For example, thermal lability and low volatility prevent gas chromatographic (GC) analysis of some compounds, while lack of a distinctive chromophore may hamper liquid chromatographic (LC) analysis. For example, the thermal lability of pyrethroid insecticides can interfere with their analysis by GC, but they can be readily quantified by immunoassay. Plant, parasite, and fungus-derived toxins are usually too large for GC, but are well suited for immunoassay. Many new, biotechnology-derived insecticides pose particularly difficult analytical problems for conventional approaches because of their large mass, but antibodies to avermecatins and Bacillus thuringiensis toxins illustrate the applicability of immunoassays for insecticides in this upcoming class of compounds.
In the area of pesticide detection, many recently developed pesticides derive their species-selectively by inhibiting specific enzymes in the target species. These substrates are often structural analogs of natural substrates, which means the chemist must determine trace levels of a pesticide in the presence of abundant amounts of the chemically-similar natural compound. With increasing public demands that pesticides not affect non-target species, the use of analogs of natural substrates will increase, and the selectivity of antibody binding offers an obvious solution to the problems of the residue analysis.
Unlike the sequential sample processing required by chromatographic methods, immunoassays allow for parallel sample processing and are inexpensive, rapid, and field-portable. In some cases immunoassays have been applied on site, resulting in additional savings in the costs and time involved in transporting samples back to a central laboratory. As screening assays, immunoassays can eliminate the need for complete work-up of negative samples, freeing the analytical laboratory to focus on the more interesting, positive samples. Ultimately, these cost savings make studies economically feasible that otherwise would be prohibitively expensive; for example, the mass screening of individual wells for drinking-water contamination, or the quantitative analysis of many epidemiological studies. They also allow the introduction of monitoring programs in rural areas where skilled personnel and sophisticated laboratories are lacking. For many applications, the analytes must be extracted from the matrix, and some level of sample preparation and cleanup is required. However, sample cleanup is usually significantly less than is required for GC/MC analysis.
Immunoassays can be combined with other techniques to exploit the advantages of both methods. For example, antibodies can be used to concentrate both the parent compound and its metabolitede from urine. Individual compounds then can be qualified by separating them by HPLC following immunoconcentration or as in the case of aflatoxin, immunopurification can then be followed by fluorescence detection, using intrinsic fluorescence of the analyte.
Applications of immunoassays include monitoring of residues in foods, in the environment, and in humans for residues of both synthetic and naturally occurring toxins. The primary motives for the development of these assays are their high sensitivity, high selectivity, portability, short analysis time, low cost, and potential for parallel processing samples. Parallel processing of samples means that immunoassays are highly applicable to mass screening studies either for monitoring regulatory compliance or for epidemiology studies. Particularly powerful analytical approaches use antibodies in conjunction with other methods, for example, the use of immunoaffinity columns to concentrate and purify the analyte before measurement by conventional means.
Examples of Particle-based Immunoassays
Microspheres or uniform particles of many sizes are used in a wide variety of modern diagnostic tests and assays. Particle-based diagnostic test qualitative/quantitative assays are usually based upon the specific interaction of antigen or analyte and antibody. Antigen or antibody can be adsorbed onto submicron sized polystyrene (PS) particles, often called "uniform latex particles". These sensitized particles then act to magnify or amplify the Antigen-Antibody reaction which takes place when a sample containing the sought molecule is mixed with the appropriately coated particles. In the classic example, a positive test results when uniformly dispersed milky appearing Ab-coated particles in a drop of water on a glass slide react with Ag in a drop of sample (whole blood, serum, urine, etc.) to cause particle agglutination (coagulation or clumping). An improvement in Latex Agglutination Tests (LATs) is the use of dyed particles which provide different contrast (dyed particles observed against a white background). They also permit some tests using samples of whole blood, if dark blue or black particles are used. As an example of the versatility of dyed particles, Wellcome Diagnostics (Dartford, Kent, England) has a Salmonella test which uses antibodies to three different antigen groups bound to three different colored particles (red, blue and green). By comparing the shade of the color of the combined agglutinated particles to a background color, one can decide which of seven combinations of Salmonella groups are present in the sample. Enzyme Immunofiltration Assays (EIFA) utilize microporous membranes as the receptor bearing solid phase and employ filtration as a means to hasten contact with the soluble sample ligand and the signal generating reagents. To prepare these bests, Ab (antibody) is adsorbed onto PS particles; the particles are caught on a filter and dried. In use: First, a sample is passed through a filter and any Ag (antigen) is caught by the Ab on the particles. Next, a second Ab-enzyme reacts with it to create an insoluble colored product which is proportional to the amount of Ag captured. The diffusion limitation of the reaction rate seen for conventional solid-phase immunoassays is minimized in EIFA. This is due to the flow of reactants through the receptor bearing membrane solid phase and the high ratio of microporous membrane surface area to liquid volume. Thus, EIFA permits rapid tests to be developed which reach completion in minutes. The antigen-antibody reactions in EIFA are visualized directly by immunostaining, in which the signal-generating conjugate yields colored spots at the reaction sites, on the membrane. The color intensity of these spots can be quantitated by reflectance photometry.
Various EIFA methods have been described for the detection of antigens by means of direct binding of sample to the membrane or by employing two antibodies in a sandwich. Detection of antibodies by permutations of this method has also been described. In the sandwich assay described by Valkirs and Barton, rapid flow followed by a short incubation period was used to give a total assay time of 5 minutes. Quantitative assays based on EIFA have reproducibility and sensitivity comparable to that of other enzyme-linked immunosorbent assay (ELISA) techniques. The EIFA system can be incorporated in a unit, which, besides, the antibody-bearing solid phase, includes an absorbing material for drawing liquid through the membrane and a waste reservoir. Because of their convenience, simplicity, and speed EIFA devices can be used in technically unsophisticated patient environments, i.e., as near patient tests. Various tests (like HCG, "strep" A, and others) using this principle have been made by Hybritech (ICON). Abbott (TestPack), Nova Nordisk A/S (NovoClone Target), and many others. Murex SUDS use liquid reagents in their tests: mixing Ab-coated particles +Ag (from sample) + second Ab-enzyme conjugate; then pouring the mixture through their filter device to capture the particles which are rinsed with enzyme substrate to form color.
Improved Dyes and Latex
Small microspheres with bright, photostable fluorescent or colored dyes have opened up new opportunities for sensitive diagnostic tests. Fluorescent latex is inexpensive and widely applicable to qualitative and quantitative immunodiagnostics. The use of fluorescent latex particles should be applicable to most, if not all of the major latex-based diagnostic test systems presently in use, including latex agglutination tests (LAT), filter separation tests (in which agglutinated particles are trapped on a filter), particle capture ELISA methods and two-particle sandwich techniques. The increased signal available from fluorescence offers the option of quantitative, as well as qualitative results, with potential sensitivity increases of over 1000-fold, compared to colorimetric methods.
Several areas for latex particles in ultra-sensitive diagnostic tests are outlined as follows:
Latex Agglutination Tests (LAT)/Latex Immunossay (LIA) PA1 Agglutination/Capture Tests & Assays (Dyed Particles) PA1 Particle Capture ELISA Tests & Assays PA1 Dyed-Particle Sandwich Tests & Assays PA1 SPRIA/SPEIA, DNA Probes (solid/liquid separation via centrifuge or magnet).
Membranes and filters have been used in a number of devices and in procedures for determining the presence of an analyte in a sample. These devices, however, are dependent on a specific sample size, typically utilize bound reagents and are dependent upon rate of diffusion of liquid sample through an absorbent material to define the time limitation s of the assay.
For example, U.S. Pat. No. 4,632,901 discloses an apparatus having a membrane or filter to which an antibody is bound and which is in liquid contact with an absorbent material. The sample size is limited by the absorbency of the material. U.S. Pat. No. 4,823,461 also discloses a membrane device having an absorbent material in fluid contact with the membrane. In this device, however, the absorbent material contacts only the periphery of the membrane thus causing liquid to diffuse transversely. The device is useful for only a limited number of sample types.
The invention is useful in detecting a broad range of analytes. U.S. Pat. Nos. 4,374,925 and 3,817,837 set out lists of analytes which are part of specific binding pairs. Analytes of particular interest include viruses, bacteria, fungi and other analytes of similar size. The examples of analytes that can be assayed with the present invention include Chlamydia, Salmonella, Bordetella, Candida and Aspergillus.
The development of a rapid on-site immunoassay system with a versatile and easy to use handheld meter provides an objective means of screening for levels of agricultural and environmental chemicals in either a remote site or laboratory setting and will enable better monitoring of the levels and movement of chemicals through the environment.
It is therefore desirable to provide an easy to handle apparatus which contains fluid samples holding analytes with a minimum chance for spillage and contamination between collection and laboratory. In addition, cells or sediment contained in the fluid have a valuable diagnostic use so that capturing the same for further testing is beneficial. In using the present invention testing can be performed quickly and accurately with minimum time. For some testing, particularly where antigens or analytes are being removed from the test fluid for a variety of tests, it is desirable to remove the antigens or analytes from the fluid so that various test procedures can be run. It is also desirable to do so with minimal exposure of laboratory personnel to the sample subject of testing. Previously this has been accomplished by a series of tests involving number of different containers and expensive laboratory equipment. Mass testing using such a series of tests is expensive, time consuming, and often unsatisfactory.